Mayaro virus detection by integrating sample preparation with isothermal amplification in portable devices.
Mayaro virus detection by integrating sample preparation with isothermal amplification in portable devices.
复制标题
通过将样品制备与便携式设备中的等温扩增相结合来检测 Mayaro 病毒。
DOI：
10.1007/s00216-023-04856-8
复制
发表时间：
2023
影响因子：
4.3
通讯作者：
Fan,ZHugh
中科院分区：
文献类型：
--
作者：
Alipanah,Morteza;Manzanas,Carlos;Hai,Xin;Lednicky,JohnA;Paniz-Mondolfi,Alberto;Morris,JGlenn;Fan,ZHugh
马亚罗病毒（MAYV）是一种新出现的蚊媒甲病毒，其引起的临床症状与基孔肯雅病毒（CHIKV）、登革热病毒（DENV）和寨卡病毒（ZIKV）引起的症状相似。为了从诊断上区分MAYV和这些病毒，我们开发了一种便携式装置，该装置将样品制备与实时逆转录环介导等温扩增（rRT-LAMP）相结合。首先，我们设计了一种针对MAYV非结构蛋白（NS 1）基因的rRT-LAMP方法，并确定了每个反应的检测限至少为10个病毒基因组当量。该测定对MAYV具有特异性，与CHIKV、DENV或ZIKV没有交叉反应。将rRT-LAMP测定与样品制备装置（SPD）整合，其中在现场进行病毒裂解和RNA富集/纯化，而不需要移液，而随后的实时扩增装置（RAD）能够在护理点（POC）进行病毒检测。使用纯化的MAYV RNA或含有活病毒的血液样品证明了我们平台的功能。我们已经使用该设备在短至13分钟内检测MAYV，检测限低至10个GE/反应。图形摘要
Mayaro virus (MAYV) is an emerging mosquito-borne alphavirus that causes clinical symptoms similar to those caused by Chikungunya virus (CHIKV), Dengue virus (DENV), and Zika virus (ZIKV). To differentiate MAYV from these viruses diagnostically, we have developed a portable device that integrates sample preparation with real-time, reverse-transcription, loop-mediated isothermal amplification (rRT-LAMP). First, we designed a rRT-LAMP assay targeting MAYV’snon-structural protein(NS1) gene and determined the limit of detection of at least 10 viral genome equivalents per reaction. The assay was specific for MAYV, without cross-reactions with CHIKV, DENV, or ZIKV. The rRT-LAMP assay was integrated with a sample preparation device (SPD) wherein virus lysis and RNA enrichment/purification were carried out on the spot, without requiring pipetting, while subsequent real-time amplification device (RAD) enables virus detection at the point of care (POC). The functions of our platform were demonstrated using purified MAYV RNA or blood samples containing viable viruses. We have used the devices for detection of MAYV in as short as 13 min, with limit of detection to as low as 10 GEs/reaction.Graphical Abstract